Human granulocyte colony-stimulating factor (hG-CSF) is a cytokine that can play a role in the proliferation and differentiation of hemopoietic precursor cells and the activation of mature neutrophilic granulocytes. Recombinant hG-CSF can be used, for example, as an injectable to i) selectively stimulate the growth of white blood cells, ii) to help reduce the incidence of infection in patients undergoing certain cancer chemotherapy, iii) for the mobilization of peripheral blood progenitor cells, and iv) for the treatment of severe chronic neutropenia.
Two forms of recombinant hG-CSF are currently available for clinical use on the market: a glycosylated form obtained by expression in mammalian cells and a non-glycosylated form synthesized in an E. coli expression system. Economical large-scale production of recombinant hG-CSF is still a great challenge with respect to biosynthesis and downstream processing because the expression efficiency of a hG-CSF gene in the E. coli expression system is low and overexpression generally results in partitioning of the expressed protein as insoluble material in inclusion bodies. Expression of hG-CSF in insoluble inclusion bodies (IBs) can require lengthy downstream process to solublize and refold the target protein. The available periplasmically expressed hG-CSF products either lack the N-terminal methionine or have alternative N-terminal sequence compared to the drug Filgrastim. There has been work reported in E. coli relating to secretion of hG-CSF in a soluble form; however, in these expression systems a peptide tag was added and tag removal upon purification required. Thus, there is a need for new methods of expressing hG-CSF comprising an N-terminal methionine (Met-G-CSF) and no sequence tag.